Relaxins are heterodimeric peptide hormones composed, in their mature form, of an A chain and a B chain linked via disulphide bridges. Human relaxins in their mature form are stabilised by three disulphide bonds, two inter-chain disulphide bonds between the A chain and B chain and one intra-chain disulphide bond between cysteine residues in the A chain.
Relaxins have been conserved through vertebrate evolution and have been characterised in a large and diverse range of vertebrate species. In particular the cysteine residues in the B and A chains responsible for the intra- and inter-chain disulphide bonds are highly conserved. Whilst in most species only two forms of relaxin have been identified (relaxin and relaxin-3), in humans three distinct forms of relaxin have been described and the genes and polypeptides characterised. These have been designated H1, H2 and H3. Homologues of H1 and H2 relaxin have been identified in other higher primates including chimpanzees, gorillas and orangutans. Differing expression patterns for H1, H2 and H3 relaxin suggest some differences in biological roles, however all three forms display similar biological activities, as determined for example by their ability to modulate (stimulate or inhibit) cAMP activity in cells expressing relaxin family receptors, and accordingly share some biological functions in common.
Relaxin-3 is predominantly expressed in the brain where it acts as a neuropeptide acting through its receptor RXFP3 as a regulator of homeostatic physiology and complex behaviours, including feeding and metabolism, and circadian arousal and sleep patterns, with strong interactions with brain stress and mood systems.
Aberrant relaxin activity and/or expression is implicated in a number of disorders and diseases and thus there exist a number of important clinical applications for relaxin and antagonists of relaxin receptors.
With the increasing therapeutic promise shown by relaxin-3 and the continued development of potential clinical applications there is also an interest in developing relaxin polypeptides that are simpler in structure than native relaxin molecules and yet which retain the ability to bind to relaxin receptors and/or retain biological activity. Simplifying the structure of therapeutic polypeptides and minimising the amino acid sequence required to impart biological activity on therapeutic polypeptides can serve to reduce the cost of polypeptide synthesis, reduce the complexity and difficulty of synthesis, and/or improve the efficiency of synthesis. Moreover, simplified, smaller molecules may exhibit improved in vivo activities and/or cellular uptake of such molecules may be improved when compared to native counterparts. In the case of relaxins, attention to date has been focused on heterodimeric polypeptides and to date single chain relaxin analogues that retain sufficient biological activity to be of therapeutic potential have not been identified.
The biological actions of relaxins are mediated through G protein coupled receptors (reviewed in Bathgate et al., 2006). To date, H1, H2 and H3 relaxins have been shown to primarily recognise and bind four receptors, RXFP1 (LGR7), RXFP2 (LGR8), RXFP3 (GPCR135) and RXFP4 (GPCR142). Interestingly, receptors RXFP1 and RXFP2 are structurally distinct from receptors RXFP3 and RXFP4, yet despite the differences there is significant cross-reactivity between different native relaxin molecules and different receptors. The endogenous receptor in the brain for H3 relaxin is RXFP3, however H3 relaxin has also been shown in cell-based systems to bind and activate both RXFP1 and RXFP4. Thus, since both RXFP1 and RXFP3 are expressed in the brain, it has been very difficult to experimentally determine the precise physiological role of relaxin-3 in the brain due to its cross-activation of RXFP1. Accordingly, there is a need for analogues of relaxin-3 that are specific for RXFP3, lacking the ability to bind and activate RXFP1 or RXFP4, or that are at least strongly selective for RXFP3.